@article{fc90c414e79347ed96ce6a33c4b4ac64,
title = "The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy",
abstract = "Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, g-tubulin ring complex (g-TuRC) has been identified, precisely how g-TuRC nucleates a MT remains poorly understood. Here, we developed a single molecule assay to directly visualize nucleation of a MT from purified Xenopus laevis g-TuRC. We reveal a high g-/ab-tubulin affinity, which facilitates assembly of a MT from g-TuRC. Whereas spontaneous nucleation requires assembly of 8 ab-tubulins, nucleation from g-TuRC occurs efficiently with a cooperativity of 4 ab-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally associated ab-tubulins drive g-TuRC into a closed conformation. NME7, TPX2, and the putative activation domain of CDK5RAP2 do not enhance g-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal g-/ab-tubulin interaction.",
author = "Akanksha Thawani and Rale, {Michael J.} and Nicolas Coudray and Gira Bhabha and Stone, {Howard A.} and Shaevitz, {Joshua W.} and Sabine Petry",
note = "Funding Information: We thank Brian Mahon, Brianna Romer and Sophie Travis for advice on collecting and processing of electron microscopy data, as well as Petry lab members for discussions. We thank David Agard and Michelle Moritz for sharing unpublished data and for discussions. This work was supported by an American Heart Association predoctoral fellowship 17PRE33660328 and a Princeton University Honorific Fellowship (both to AT), a Howard Hughes Medical Institute Gilliam fellowship and a National Science Foundation graduate research fellowship (both to MJR), NIGMS R00GM112982 (to GB), NIH New Innovator Award 1DP2GM123493, Pew Scholars Program in the Biomedical Sciences 00027340, David and Lucile Packard Foundation 2014–40376 (all to SP), and the Center for the Physics of Biological Function sponsored by the National Science Foundation grant PHY-1734030. Funding Information: We thank Brian Mahon, Brianna Romer and Sophie Travis for advice on collecting and processing of electron microscopy data, as well as Petry lab members for discussions. We thank David Agard and Michelle Moritz for sharing unpublished data and for discussions. This work was supported by an American Heart Association predoctoral fellowship 17PRE33660328 and a Princeton University Honorific Fellowship (both to AT), a Howard Hughes Medical Institute Gilliam fellowship and a National Science Foundation graduate research fellowship (both to MJR), NIGMS R00GM112982 (to GB), NIH New Innovator Award 1DP2GM123493, Pew Scholars Program in the Biomedical Sciences 00027340, David and Lucile Packard Foundation 2014?40376 (all to SP), and the Center for the Physics of Biological Function sponsored by the National Science Foundation grant PHY-1734030. American Heart Association 17PRE33660328 Akanksha Thawani Princeton University Charlotte Elizabeth Procter Honorific Fellowship Akanksha Thawani Howard Hughes Medical Insti- tute Gilliam fellowship Michael J Rale National Science Foundation Graduate Student Fellowship Michael J Rale National Institute of GeneralMedical Sciences R00GM112982 Gira Bhabha National Institute of General Medical Sciences 1DP2GM123493 Sabine Petry Pew Charitable Trusts 00027340 Sabine Petry David and Lucile Packard Foundation 2014-40376 Sabine Petry National Science Foundation PHY-1734030 Joshua W Shaevitz. Publisher Copyright: {\textcopyright} Thawani et al.",
year = "2020",
month = jun,
doi = "10.7554/eLife.54253",
language = "English (US)",
volume = "9",
pages = "1--34",
journal = "eLife",
issn = "2050-084X",
publisher = "eLife Sciences Publications",
}